A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.
A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.
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DOI：
10.3389/fpls.2014.00557
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发表时间：
2014
影响因子：
5.6
通讯作者：
Pfannschmidt T
中科院分区：
文献类型：
--
作者：
Schröter Y;Steiner S;Weisheit W;Mittag M;Pfannschmidt T
植物子叶是一种在叶绿体基因表达方面特别活跃的组织，以便从原叶绿体发育成功能叶绿体，原叶绿体是植物胚胎中的叶绿体前体阶段。因此，子叶是研究与叶绿体基因表达有关的蛋白质复合体的组成、功能和调控的理想材料。在这里，我们提出了一项初步研究，使用肝素-琼脂糖和磷酸纤维素层析结合等电聚焦和变性十二烷基硫酸钠凝胶电泳法(双向凝胶电泳法)来研究从子叶中纯化的芥菜叶绿体的核酸结合亚蛋白质组。我们描述了从这样的样品中获得的数百个蛋白质点的高分辨率生物化学纯化的技术要求。随后，从经过胰酶处理的切点中分离出的多肽进行了质谱分析，在180个不同的点中鉴定出了58种不同的蛋白质。我们的分析表明，参与转录和翻译的蛋白质高度丰富，此外，这个叶绿体蛋白亚组分存在大量的翻译后修饰。本研究为进一步分析低丰度基因表达相关蛋白提供了一系列与基因表达及其调控相关的芥菜叶绿体蛋白，并为进一步分析低丰度基因表达相关蛋白提供了合适的纯化策略。
Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.
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影响因子：
7.4
作者：
BAUMGARTNER, BJ;RAPP, JC;MULLET, JE
通讯作者：
MULLET, JE
影响因子：
5.1
作者：
Baginsky, S;Tiller, K;Link, G
通讯作者：
Link, G
影响因子：
5.1
作者：
Baginsky, S;Tiller, K;Link, G
通讯作者：
Link, G
影响因子：
4.5
作者：
Cavaillé, J;Chetouani, F;Bachellerie, JP
通讯作者：
Bachellerie, JP
DOI：
10.1073/pnas.182432999
发表时间：
2002-09-17
影响因子：
11.1
作者：
Martin, W;Rujan, T;Penny, D
通讯作者：
Penny, D